KR20220074132A - Dermal fibroblasts exhibiting tendon regeneration effect and uses thereof - Google Patents

Abstract

The present invention relates to human skin-derived fibroblasts exhibiting a tendon regeneration effect and a pharmaceutical composition for tendon regeneration and treatment comprising the same as an active ingredient, wherein the allogeneic skin-derived fibroblasts are cellularly morphologically similar to tendon cells, and fibers It has a high level of expression of the blast marker vimentin, a large amount of extracellular matrix protein, and can increase collagen synthesis by acting on TGF-β and ERK signaling mechanisms, so that damaged tendons can be efficiently regenerated.

Description

Skin-derived fibroblasts exhibiting tendon regeneration effect and uses thereof

The present invention relates to skin-derived fibroblasts exhibiting a tendon regeneration effect and a pharmaceutical composition for tendon regeneration and treatment comprising the same as an active ingredient.

Tendon is a connective tissue that connects muscle and bone and transmits the force generated from the muscle to the bone to cause joint movement. Extra cellular matrix such as tenocyte and collagen , ECM). Tendon cells are tendon-specific fibroblasts that play a role in the production and maintenance of the tendon matrix. Tendon damage due to trauma is common in exercise, work, and daily life.

A rotator cuff tear is a tendon (tendon) of the four muscles that surround the shoulder blade and help the arm move freely: supraspinatus, infraspinatus, teres minor, and subscapularis. , tendon) is partially or completely ruptured due to causes such as aging. Rotator cuff tears account for about 70% of shoulder lesions, and the incidence is increasing with aging and the increase in the sports population. Most of the treatments up to now are surgical sutures, but the rate of re-rupture after suturing is high, so there is a need to develop alternative treatments.

Although there are autologous cell therapy using tendon cells as a similar cell therapy commercially available to date, there is a disadvantage in that it is necessary to accompany surgery to collect the patient's normal tendon tissue to obtain raw cells for cell therapy.

On the other hand, if fibroblasts can be used as therapeutic agents, fibroblasts can be easily harvested in a relatively non-invasive way, and can be manufactured in a ready-made form with quality standards secured so that they can be used immediately when necessary. It can solve the problems of cell therapy products using autologous tendon cells. Therefore, there is a need to develop a therapeutic agent for tendon damage using fibroblasts in addition to allogeneic tendon cells.

1. Republic of Korea Patent No. 10-2091442 2. US Patent Publication No. 2013-0295061

In order to solve the above problems, the present inventors tried to develop a therapeutic agent for tendon damage using fibroblasts, derived from human skin, and compared to vimentin, collagen I, collagen III, collagen V, fibronectin and elastin compared to tendon cells. The present invention was completed by confirming that fibroblasts with increased expression of one or more selected from the group consisting of

In order to solve the above problems, one object of the present invention is

Fibroblasts derived from the skin and having a tendon regeneration effect characterized by increased expression of one or more proteins selected from the group consisting of vimentin, collagen I, collagen III, collagen V, fibronectin and elastin compared to tendon cells ( skin-derived fibroblasts).

As used herein, "fibroblast" means a mesodermal cell with high diversity found in various tissues, and according to the US National Institutes of Health database MeSH, the definition is 'connective tissue that abundantly secretes collagen and high molecular substances' cells (connective tissue cells which secrete an extracellular matrix rich in collagen and other macromolecules).

As used herein, the term "tendon" is a connective tissue that connects muscles and bones and transfers the force generated from the muscles to the bones to cause joint movement. Type I collagen accounts for 85-95% of the tendon weight. , Type III collagen accounts for less than about 5%, and proteoglycan accounts for less than about 5%. In addition, fibronectin, elastin, and the like provide a solid cell scaffold in the tissue.

The present inventors used fibroblasts capable of obtaining autologous or allogenic cells in a relatively non-invasive manner in order to solve the problem of cell therapy using autologous tendon cells. Specifically, the normal skin tissue isolated from the donor was minced, and only the fibroblasts were isolated by decomposing the cell matrix. The donor may be a person evaluated as being suitable for cell bank construction through a suitability assessment, and may not be limited to a specific donor. Accordingly, the skin-derived fibroblasts of the present invention may be autologous or allogeneic. Allogeneic fibroblasts refer to fibroblasts originating from another person.

Fibroblasts isolated from the donor start primary culture at 37° C. and 10% CO ₂ conditions, and unlike normal cell culture, 10% CO ₂ was used for cell culture to maintain the pH of the cell culture medium at 7.4. When the cells in the culture dish reached a pre-confluent density of about 70% to 80%, subculture was performed, and a master cell bank at passage 2 and a cell bank for production at passage 6 were established.

Therefore, according to one embodiment of the present invention, the skin-derived fibroblasts are subcultured for 2 or more passages, and may preferably be cultured for 6 or more passages, and passaged in a medium of pH 6.5 to pH 8.0.

In addition, according to one embodiment of the present invention, the skin-derived fibroblasts have a cell proliferation level per passage in the range of 2.5 to 5.0, and this number is maintained at a relatively constant level even if the passage culture is continued. On the other hand, in human tendon cells, the level of cell proliferation significantly decreased when subculture was continued ( FIG. 2 ). Here, one passage refers to cells cultured for about 6 days at a pre-confluent density after seeding the cells in one culture dish.

According to one embodiment of the present invention, the skin-derived fibroblasts may have increased expression of cytokines and signaling substances reported to be involved in extracellular matrix formation and tissue regeneration promotion. Specifically, compared with tendon cells, TGF-β (transforming growth factor-β) secretion is increased, and p-ERK 1/2 (phosphorylated extracellular-signal regulated kinase 1/) involved in ERK (Extracellular-signal regulated kinase) signaling 2) expression and collagen I expression are also increased.

In addition, the skin-derived fibroblasts of the present invention have a cell viability of about 70% or more even when thawed after long-term storage, and the extracellular matrix collagen is 0.10 μg or more per unit cell number 10 ⁶ , so the quality of the cells is also remarkably excellent. .

Accordingly, another object of the present invention is to provide a pharmaceutical composition for the improvement or treatment of a tendon disease comprising the fibroblasts having the tendon regeneration effect as an active ingredient.

According to one embodiment of the present invention, the tendon disease may be selected from the group consisting of rotator cuff tear, Achilles tendon disease, tendinitis, tendon injury, and tendon dissection, preferably a rotator cuff tear.

The present inventors confirmed that the muscle tissue was regenerated to a level similar to that of the normal group without a rotator cuff tear as a result of injecting fibroblasts having a tendon regeneration effect into the rotator cuff tear animal model (FIG. 8).

In addition, as a result of treating tendon cells with a lysate of fibroblasts having a tendon regeneration effect to confirm the mechanism of tendon regeneration, the expression of p-ERK 1/2, which is involved in ERK signaling, was remarkably increased, and collagen I It was confirmed that expression also increased (FIG. 9).

The pharmaceutical composition for improving or treating the tendon disease may further include adjuvants such as hyaluronic acid, alginate, and fibrin, which are polymer carriers capable of enhancing drug efficacy. Such a polymer carrier must be biocompatible, and it can help the cell delivery, improve the physical properties without affecting the cell viability, and enhance the drug efficacy.

In addition, the pharmaceutical composition for improving or treating dry disease may further contain DMSO (dimethyl sulfoxide), which is a cell cryoprotectant, and may be included at a concentration of 2% to 15% of the cell freezing medium. DMSO prevents dehydration by changing the concentration of the impermeable extracellular solution during ice formation at the time of cell freezing.

In addition, the pharmaceutical composition for the improvement or treatment of the tendon disease may further include a pharmaceutically acceptable carrier in addition to the active ingredient. Pharmaceutically acceptable carriers included in the composition are those commonly used in the preparation of formulations, and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystals. sex cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, and the like.

The pharmaceutical composition for improving or treating the tendon disease is preferably administered parenterally. In the case of parenteral administration, it may be administered by subcutaneous injection, intramuscular injection, intraperitoneal injection, endothelial administration, topical administration, etc., and may be directly injected into the affected area.

A suitable dosage of the pharmaceutical composition for the improvement or treatment of the tendon disease is within the range of 100-100,000,000 (10 ² to 10 ⁸ ) cells/kg based on an adult. The term “pharmaceutically effective amount” means an amount sufficient to prevent or treat tendon damage.

The composition may be prepared in a unit dose form by formulating using a pharmaceutically acceptable carrier and/or excipient according to a method readily practiced by a person skilled in the art, or may be prepared by internalizing in a multi-dose container. In addition, the composition may be administered as an individual therapeutic agent or may be administered in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. In addition, it may be administered once or additionally as needed, used in combination with a surgical operation such as suturing, or administered alone.

A pharmaceutical composition for improving or treating a tendon disease comprising fibroblasts having the tendon regeneration effect as an active ingredient may be provided in the form of a cell therapeutic agent. 'Cell therapy product' means 'in order to restore the tissue and function of cells, treatment and diagnosis through a series of actions such as proliferating and selecting living autologous, allogeneic or xenogeneic cells in vitro or changing the biological characteristics of cells in other ways and drugs used for prophylactic purposes.

Allogeneic skin-derived fibroblasts having a tendon regeneration effect according to an embodiment of the present invention are cytomorphologically similar to tendon cells, have a high expression level of vimentin, a fibroblast marker, express a large amount of extracellular matrix protein, and TGF- It can increase collagen synthesis by acting on the β and ERK signaling mechanism, so that damaged tendons can be efficiently regenerated.

1 is a schematic diagram of constructing a human-derived allogeneic dermal fibroblast bank with skin-derived fibroblasts according to an embodiment of the present invention.
2 is a result of confirming the cell proliferation level (population doubling level, PDL) according to the subculture of human-derived tendon cells and skin-derived fibroblasts.
3 is a result of confirming in vitro cultured tendon cells and skin-derived fibroblasts of the master and manufacturing cell bank with a phase-contrast microscope.
Figure 4 is the result of confirming the expression level of vimentin in the in vitro cultured tendon cells and skin-derived fibroblasts of the cell bank for production.
5 is a result of confirming the expression levels of keratinocyte markers keratin (K14) and pan-cytokeratin (Pan-cytokeratin) in skin-derived fibroblasts of the cell bank for manufacturing.
6 is a result of confirming the ratio of non-mentin-expressing cells in the skin-derived fibroblasts of the cell bank for production by flow cytometry.
7 is a result of confirming the expression level of the extracellular matrix protein in the in vitro cultured tendon cells and the skin-derived fibroblasts of the cell bank for production by fluorescence microscopy (A) and Western blot (B).
8 is a result of histological analysis of the degree of recovery of tendon tissue after injection of skin-derived fibroblasts into an animal model of rotator cuff tear.
In FIG. 9, A is the result of confirming the level of TGF-β secreted from the skin-derived fibroblasts, and B is the result of confirming the expression change of the signaling molecule after treating the tendon cells with the cell lysate of the skin-derived fibroblasts.

Hereinafter, one or more specific examples will be described in more detail through examples. However, these examples are for illustrative purposes of one or more embodiments, and the scope of the present invention is not limited to these examples.

Example 1: Culture of skin-derived fibroblasts

The normal skin tissue isolated from the donor was minced and decomposed to isolate only fibroblasts. The isolated fibroblasts were subjected to primary culture in Dulbecco's Modified Eagle's medium (DMEM)/F12 (Invitrogen, USA) containing 10% fetal bovine serum (FBS), and the culture condition was 37°C. , a humid environment of 10% CO ₂ was maintained. After subculture at intervals of about 5 to 6 days, a Master Cell Bank (MCB) was constructed in passage 2. Thereafter, additional subcultures were performed at intervals of about 5 to 6 days, and a large amount of Working Cell Bank (WCB) was secured at passage 6 (FIG. 1). Cell subculture was performed by separating the cells with trypsin-EDTA when the cells in the culture dish were about 70-80% full. In most in vitro cell cultures, 5% CO ₂ is used as a gas composition, but in the present invention, 10% CO ₂ was used to accurately maintain the pH of the medium at 7.4.

The cell bank was frozen and stored as follows. First, cells of passages 2 to 10 were mixed with a freezing medium containing less than 10% of dimethyl sulfoxide (DMSO), a cryoprotectant for cells. After that, 1-10x10 ⁶ cells in a suspended state were dispensed in a glass-formed ampoule, and the cells were frozen while gradually dropping the temperature with isopropanol, and finally stored for a long time in a freezer or liquid nitrogen below -15°C.

Hereinafter, the human skin-derived fibroblasts obtained in this example will be referred to as "skin-derived fibroblasts".

Example 2: Characterization of skin-derived fibroblasts

2-1. Proliferative and Morphological Characteristics of Skin-derived Fibroblasts

The skin-derived fibroblasts obtained in Example 1 were cultured from passage 2 to passage 10 under the same culture conditions, and the number of cells during initial plating and the total number of cells after obtaining the cells were counted to determine the doubling time of the cells. Calculated. As a result, it took an average of 30 hours from passages 2 to 10, and showed similar values for two different media.

In addition, as a result of confirming the cell proliferation level (population doubling level, PDL) according to Equation 1 below, 2.7 to 4.5 per passage of the skin-derived fibroblasts obtained in Example 1 were calculated ( FIG. 2 , lower graph; N= 3, mean ± standard deviation). This result is in contrast to that tendon cells have a short cell lifespan when cultured in vitro (FIG. 2, upper graph; N=3, mean ± standard deviation), and is one of the important advantages of using fibroblasts as a cell therapy agent.

[Equation 1]

PDL = (log (NH)-log (NI))/log 2

NH: number of finally obtained cells; NI: Initial plating cell count.

In addition, as a result of confirming the morphology of skin-derived fibroblasts in the master and manufacturing cell bank prepared in Example 1, the skin-derived fibroblasts of the present invention maintain the fibroblast-specific spindle-shaped morphology similar to tendon cells. It was confirmed that (FIG. 3).

2-2. Confirmation of vimentin expression

The cultured tendon cells and skin-derived fibroblasts were treated with a primary antibody against the fibroblast-labeled protein vimentin and a green fluorescent material (FITC)-binding secondary antibody, followed by observation under a fluorescence microscope. Cell nuclei were stained with DAPI (4,6-diamidino-2-phenylindole).

As a result, it was confirmed that the skin-derived fibroblasts of the cell bank for manufacturing showed unique characteristics of fibroblasts and expressed vimentin at a level similar to or higher than that of tendon cells (FIG. 4).

2-3. Homogeneity check

In order to confirm the mixing and homogeneity in the skin-derived fibroblasts of the manufacturing cell bank, keratinocyte markers keratin (K14) and pan-cytokeratin (Pan-cytokeratin) antibodies were used to stain the cells, followed by fluorescence microscopy and flow cytometry. Analysis was performed. Cell nuclei were stained with DAPI.

As a result, no keratinocyte marker was detected in the qualitative observation under a fluorescence microscope (FIG. 5). In addition, in flow cytometry, which is a quantitative analysis, the average number of cells classified as non-mentin-expressing fibroblast markers was 98.5%, confirming that most of the fibroblast characteristics of skin-derived fibroblasts constituting the manufacturing cell bank were confirmed (Fig. 6).

2-4. Identification of extracellular matrix proteins

The expression level of collagen, which is a representative extracellular matrix, was confirmed in the skin-derived fibroblasts of the manufacturing cell bank. Skin-derived fibroblasts were treated with each primary antibody and a green fluorescent material (FITC)-binding secondary antibody against extracellular matrix protein, and then observed under a fluorescence microscope.

As a result, it was found that the expression levels of collagen I, III and IV, fibronectin and elastin were very high in skin-derived fibroblasts (FIG. 7).

Example 3: Confirmation of quality of skin-derived fibroblasts

After thawing the skin-derived fibroblasts of the cryopreserved cell bank, the quality was confirmed by culturing again. As a result, the thawing yield and cell viability of the re-cultured skin-derived fibroblasts were excellent, confirming that the cell quality was maintained regardless of the freezing period (Table 1).

Batch Quality Analysis of Manufacturing Cell Banks Freezing period (days) Thawing yield (%) Cell viability (%) Amount of collagen per 10 ⁶ cells (μg) One 45 100 71 0.14 2 136 88.7 77 0.33 3 1830 98.5 72 0.32

In addition, as a result of performing an adventitious virus negative test and microbiological residual test that meet the cell therapy approval criteria of the Ministry of Food and Drug Safety, it was confirmed that no adventitious viruses and microorganisms remained in the cell bank.

Experimental Example 1: Confirmation of rotator cuff tear treatment efficacy

1-1. Preclinical efficacy

The efficacy of the manufacturing cell bank for treating rotator cuff tear of skin-derived fibroblasts was confirmed as follows.

Bilateral supraspinatus tears were performed on the shoulders of 20-week-old New Zealand White rabbits, and after 6 weeks, saline was injected into the control group and 10 ⁷ skin-derived fibroblasts were injected into the experimental group, followed by sutures. After 12 weeks had elapsed after suturing, it was confirmed that there were no abnormalities in the rabbit's weight and other abnormalities, the rabbit was sacrificed, the shoulder tendons were removed, and a paraffin block was prepared by decalcification. Paraffin blocks were stained with Masson's trichrome and analyzed histologically.

As a result of the analysis, in the experimental group injected with skin-derived fibroblasts compared to the control group injected with saline, an aligned and dense tendon tissue arrangement at a level similar to that of the normal group without rotator cuff tear was observed (FIG. 8). In FIG. 8 , parts stained in blue are collagen, and parts stained in red are muscle, cytoplasm, and keratin.

1-2. Confirmation of therapeutic efficacy mechanism

In order to confirm the mechanism of efficacy in treating rotator cuff tear in skin-derived fibroblasts, cytokines and signaling substances reported to be involved in extracellular matrix formation and tissue regeneration promotion were analyzed.

As a result of culturing human-derived tendon cells and skin-derived fibroblasts of the cell bank for manufacturing, the level of transforming growth factor-β (TGF-β) was quantified by ELISA (Enzyme-linked immunosorbent assay). As a result, skin-derived fibroblasts (HF) were By secreting 692 pg of TGF-β per 1 x 10 ⁶ cells, the secretion amount was higher than that of tendon cells (HT) ( FIG. 9A ).

In addition, after treating the cell lysate of skin-derived fibroblasts in tendon cells, cell signaling molecules involved in signal transduction were confirmed by Western blot. As a result, the expression of p-ERK 1/2 (phosphorylated extracellular-signal regulated kinase 1/2) was remarkably increased in the experimental group (HF lysate) treated with the skin-derived fibroblast lysate compared to the control group, and the expression of collagen I It was also found to increase ( FIG. 9B ). These results suggest that the skin-derived fibroblasts of the present invention increase collagen I expression by ERK signaling and, as a result, show a tendon regeneration effect (FIG. 9).

Claims (10)

derived from the skin,
A fibroblast having a tendon regeneration effect, wherein the expression of one or more proteins selected from the group consisting of vimentin, collagen I, collagen III, collagen V, fibronectin and elastin is increased compared to tendon cells. According to claim 1, wherein the fibroblasts will be autologous or allogeneic, fibroblasts having a tendon regeneration effect. According to claim 1, wherein the fibroblasts are subcultured for two or more passages, fibroblasts having a tendon regeneration effect. According to claim 3, wherein the fibroblasts will be passaged for one or more passages in a medium of pH 6.5 to pH 8.0, fibroblasts having a tendon regeneration effect. According to claim 1, wherein the fibroblasts have a cell proliferation level per passage (population doubling level) in the range of 2.5 to 5.0, fibroblasts having a tendon regeneration effect. According to claim 1, wherein the fibroblasts have an increased TGF-β (transforming growth factor-β) secretion compared to the tendon cells, fibroblasts having a tendon regeneration effect. According to claim 1, wherein the fibroblasts activate the ERK (extracellular-signal regulated kinase) signaling pathway, fibroblasts having a tendon regeneration effect. A pharmaceutical composition for improving or treating a tendon disease comprising the fibroblasts having the tendon regeneration effect of claim 1 as an active ingredient. According to claim 8, wherein the tendon disease is selected from the group consisting of rotator cuff tear, Achilles tendon disease, tendinitis, tendon damage and tendon detachment, a pharmaceutical composition for improving or treating a tendon disease. According to claim 8, wherein the composition further comprises a polymer carrier selected from the group consisting of hyaluronic acid (hyaluronic acid), alginate (alginate) and fibrin (fibrin), a pharmaceutical composition for the improvement or treatment of tendon diseases.

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